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DNA Sequencing Scenario

DNA Sequencing Scenario. DNA replication and chain termination. ddTTP No OH group = chain termination at T residue. dATP. O H. Ö H. H. 5 ´. T. P. P. P. P. P. P. P. P. P. P. C. T. A. G. A. A. G. T. C. T. A. T. C. P. P. P. P. P. P. P. P. 3 ´.

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DNA Sequencing Scenario

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  1. DNA Sequencing Scenario

  2. DNA replication and chain termination ddTTP No OH group = chain termination at T residue dATP OH ÖH H 5´ T P P P P P P P P P P C T A G A A G T C T A T C P P P P P P P P 3´ Adapted from Lehninger Principles of Biochemistry 4th Edition Primer strand The 3´ hydroxyl (OH) group interacts with the phosphate group on the incoming nuclueotide to form a phosphodiester bond Template strand

  3. Nucleosides & Nucleotides Taken from: http://en.wikipedia.org/wiki/Nucleotide Nucleoside = a heterocyclic base (A, G, C, U or T) and a five carbon sugar (pentose) either ribose for RNA or deoxyribose for DNA Nucleotide = a heterocyclic base and a pentose sugar with one or more phosphate groups (mono-, di- or tri-phosphate). Nucleotides are the monomers of nucleic acids, with three or more bonding together in order to form a nucleic acid.

  4. Base O H H H H O- O- O- O- O- O- O- O- O- OH OH P P P -O O P O P O CH2 -O -O O O P P O O P P O O CH2 CH2 O O O O O O O O O Base O H H H H OH H Base O H H H H H H Nucleotide structure continued Nucleoside tri-phosphate (NTP) With a ribose sugar (OH at C2 & C3) used to synthesise RNA. Deoxynucleoside tri-phosphate (dNTP) With a deoxyribose sugar(OH at C3) used to synthesise DNA. Dideoxynucleoside tri-phosphate (ddNTP) With a dideoxyribose sugar(no OH groups) used for the Sanger method (dideoxy) sequencing.

  5. phosphodiester bond http://en.wikipedia.org/wiki/Phosphodiester_bond Nucleotides are joined together by phosphodiester bonds Phosphodiester bonds require a hydroxyl group (OH) to be present on the 3´ carbon to react with a phosphate on in 5´ carbon of the sugar residue of the next nucleotide. If no 3´ hydroxyl group is available no further nucleotides can join the DNA strand and the chain terminates.This is how dideoxynucleotides prevent chain elongation – they lack a 3´ OH group on the sugar.

  6. Components of the reaction mix • DNA template • DNA primer • DNA polymerase -Taq polymerase • Mixture of deoxy (dNTPs) & fluorescently labelled dideoxy nucleotides (ddNTPs) Computer analysis – base calling Outline of Fluorescent Dideoxy DNA Sequencing 20 – 35 cycles Apply to a capillary gel & subject to electrophoresis The fragments are detected after excitation of the labelled ddNTPs by a laser Fluorescent peaks and bases are visualised and analysed

  7. T TC PRIMER TCAGAT PRIMER PRIMER PRIMER PRIMER PRIMER PRIMER PRIMER TCA TCAG TCAGATAG TCAGA TCAGATA The sequencing reaction continued Components in the reaction mix Taq polymerase OH 5´ PRIMER + dATP, dCTP, dGTP, dTTP, + ddATP, ddCTP, ddGTP, ddTTP AGTCTATATC 3´ TEMPLATE Termination products obtained Remember that the sequence obtained with be complementary to the template sequence! Therefore our new 5´  3´ complementary sequence to our original template sequence reads TCAGATAG.

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